Chronic kidney disease (CKD) is a major health problem in the US, with prevalence of >10% among all adults. Diabetes, hypertension, obesity and heart disease are common risk factors. CKD is currently managed with limited therapeutic options that act systemically and there are currently no drugs that directly and selectively target the kidney cells. Podocytes are specialized epithelial cells that are central for kidney function. Podocytes form the filtration barrier in the kidney and are a validated cellular target for proteinuric kidney diseases. Yet, rational development of podocyte protective therapeutics has been greatly hampered by lack of screening assays. We recently developed a novel podocyte cell-based screening assay and optimized it for use in 96-well plates. Our image-based assay showed Z' values of 0.46 and 0.44 in two independent primary assay readouts and a Z' > 0.65 for a multi-parametric classifier assay readout. Upon screening of a bioactive library of >2100 diverse chemicals, we identified 24 hits, validated them in secondary and functional in vitro assays and validated one select hit in an established in vivo model of proteinuric kidney disease. Analysis of specific molecular markers of injury also provided us with a potential mechanism of action of the novel hit compound. These data suggest that our novel assay could be a basis for rational chemical library profiling on podocytes. Thus, here, we propose that our cell based phenotypic assay could be further miniaturized and applied in an HTS environment to identify novel small molecules that protect podocytes from injury. Additionally, the identified hit could be validated in available downstream in vitro and in vivo assays. Our long-term goal is to develop novel podocyte- protective compounds for the treatment of a variety of proteinuric kidney diseases in humans. Our three proposed aims include: 1) Implementation and miniaturization of a newly developed podocyte cell-based assay for use in an HTS environment, 2) Execution of HTS campaign using the novel assay followed by confirmation and prioritization of the hit compounds, and 3) Validation of identified hits using in vitro and in vivo assays. We have all the in vitro and in vivo assays in place and validated in the laboratory. We have also assembled a team of experts at Rush and at Sanford-Burnham to successfully implement our proposed aims. We expect that our proposed studies, if successful, will lead to identification of novel chemical probes and compounds that directly target podocytes and protect them from injury. We also expect that the identified compounds could be further developed into therapeutics for treating proteinuric kidney disease in humans in the future, thus improving lives of patients with CKD.